Complete mitochondrial genome of Belligobio pengxianensis (Cypriniformes: Gobionidae)

Abstract Belligobio pengxianensis is a small fish endemic to the upper Yangtze River of China. In this study, the complete mitochondrial genome of B. pengxianensis is determined for the first time, and it should become a reference sequence to aid in species identification, biodiversity monitoring and conservation. The mitogenome has overall length of 16,610 bp and AT content of 55.23%, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one non-coding control region. The results of phylogenetic analyses show that B. pengxianensis is nested within the genus Hemibarbus.

B. pengxianensis ( Figure 1) is a small (standard length of less than 13 cm) fish (Yue 1998;Li 2020). In the very few studies (Ding 1994;Wang et al. 2017), it was considered to be rare, on the basis of its known records only in the Jianjiang River (a tributary of the upper Yangtze River). In a recent study (Li 2020), B. pengxianensis has been found to also be distributed in the Fujiang River (a tributary of the upper Yangtze River) based on molecular species delimitation using the mitochondrial cytochrome b gene and eight nuclear loci. Li (2020) has also indicated that B. pengxianensis and B. nummifer could not be differentiated by previously defined diagnostic characteristics (Yue 1998), such that prior records on B. nummifer in multiple tributaries of the upper Yangtze River (Ding 1994;Lei et al. 2015;Chen et al. 2017) might be due to misidentification of B. pengxianensis.
Therefore, the ambiguities in species identification between B. pengxianensis and B. nummifer, based on morphological characteristics, require molecular evidence to delimit species boundaries.
In this study, the complete mitochondrial genome of B. pengxianensis is determined for the first time. The mitogenome should become a reference sequence to aid in molecular species identification, biodiversity monitoring and conservation, and is also useful in clarifying phylogenetic position of B. pengxianensis.

Materials
The specimen of B. pengxianensis was collected in a dead state from Tongji Town, Pengzhou City, Sichuan Province, China (31.1611 N, 103.8394 E) on 12 September 2015. We obtained the sample from the fish catches of a local fisherman. It was placed in the Zoological Museum of Fudan University under the voucher number FDZM-BPPZ20150912-01 (Cuizhang Fu, czfu@fudan.edu.cn).

Methods
Total genomic DNA was extracted from muscle tissues using a high-salt method (Miller et al. 1988). Thirteen primer pairs were used to amplify DNA fragments, with the same PCR system and settings as Chai and Fu (2020). The forward primer of Primer4, reverse primer of Primer6 as well as primers of Primer12 were newly designed in this study (supplemental Table S1 in Appendix I), while the remaining primers have been previously published (Chai and Fu, 2020). A total of 13 segments were obtained through Sanger sequencing using the ABI 3730 platform (Applied Biosystems, Foster City, USA). Using a reference genome from B. nummifer (KJ413052; Wang et al. 2016), contiguous and overlapping sequences were assembled into mitochondrial genome by Sequencher 5.4 (Gene Codes, Ann Arbor, MI), based on the default parameters. Then, the assembled mitogenome was annotated by MitoAnnotator (Iwasaki et al. 2013; http://mitofish.aori.u-tokyo.ac.jp/annotation/input.html). The genome map was drawn using the CGView Server (Grant and Stothard 2008).

Results
A reference Image of B. pengxianensis is provided in Figure 1. B. pengxianensis is mainly distinguished from other species in the Gobionidae by the combination of three morphological characteristics, as follows: Three row teeth, soft and slim of the last simple dorsal ray, and the scales on the chest hidden under the skin (Yue 1998).
The complete mitochondrial genome of B. pengxianensis (MW080628) was assembled from PCR products of 13 segments (supplemental Appendixes I and II). This mitogenome is 16,610 bp in length with the AT content of 55.23%. The circular mitogenome consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one non-coding control region (Figure 2). The characteristics of the mitogenome are provided in Table 1. The protein-coding genes start with ATG/GTG and end with TAA/TAG/TA-/T-. Two conserved blocks of the control region (5 0 -CAAACCCCCCTACCCCCT-3 0 and 5 0 -TGTCAAACCCCGAAACCA-3 0 ) named CSB-II and CSB-III by Lee et al. (1995), are located in 16, 370-16,387 bp and 16,412-16,429 bp, respectively. The reconstructed phylogenetic trees display consistent topologies under the analyses of ML and BI using 13 proteincoding genes (Figure 3) or complete mitochondrial genomes (supplemental Figure S2 in Appendix III). The results show that B. pengxianensis and B. nummifer don't form the sister relationships, and all of them are nested within the genus Hemibarbus with high support values.

Discussion and conclusion
In this study, we report the mitochondrial genome of B. pengxianensis for the first time. This mitogenome displays the same patterns in the gene compositions, gene arrangements, A þ T bias of base compositions, the use of start or stop codon, and conserved sequence blocks of the control region as B. nummifer (Wang et al. 2016) and other gobionid species (see, e.g. Kim et al. 2009;Li et al. 2012;Chai and Fu 2020;Tian et al. 2022). The phylogenetic nesting of B. pengxianensis and B. nummifer in the genus Hemibarbus was revealed in our phylogeny, bringing into question the validity of the genus Belligobio. In summary, the new mitogenome of B. pengxianensis could contribute to reconstructing inter-specific relationships among species of the genera Hemibarbus and Belligobio, and is also very useful as reference molecular information to support species identification, biodiversity monitoring and conservation in the future. Author contributions FCZ conceived this study; XB and LMY conducted the experiments; XB and NXM analyzed the data, XB wrote the drafting of the paper; FCZ and NXM revised it critically, and that all authors agree to be accountable for all aspects of the work.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/ under the accession no.MW080628. Sanger sequencing results are provided in supplementary materials.